表皮生长因子受体(EGFR)的细胞内信号传导

表皮生长因子受体（EGFR）是一种存在于细胞表面的多功能跨膜蛋白分子,具有酪氨酸蛋白激酶活性,EGFR与配体结合后启动细胞内信号传导通路,不同的通路之间存在交叉对话（Cross-talks）共同完成细胞生理功能.对EGFR的深入研究,不仅可阐明细胞生长和发育等重要的生命过程,而且在医药和工业上也将有广泛的应用.     

血管内皮生长因子受体1酪氨酸激酶的克隆、表达和功能

血管内皮生长因子受体 1(Flt 1)在血管生成过程中起着重要的作用。Flt 1胞内域的酪氨酸激酶直接参与了VEGF与Flt 1结合后的胞内信号转导途径。在原核系统中表达得到具有酶活性的Flt 1酪氨酸激酶融合蛋白 ,并进行了初步的性质研究。利用逆转录PCR技术从人肝癌组织总RNA中得到Flt 1酪氨酸激酶区的cDNA ,将其克隆到表达载体质粒 pGEX KG中 ,并在大肠杆菌BL2 1(DE3) pLysS中表达、纯化 ,得到可溶的Flt 1酪氨酸激酶融合蛋白 (GST F)。虽然GST F不包含目前已知的磷酸化位点 ,但研究表明GST F能够进行自磷酸化反应 ,并且其活性需要镁离子或锰离子的参与。同时发现GST F能够磷酸化合成底物 polyE4Y ,而不能作用于MBP和Src相关肽。底物磷酸化时最适的镁离子和锰离子浓度分别为 15mmol/L和 0 .5mmol/L。GST F为寻找抗肿瘤药物提供了一个有效工具     

Receptor Level Mechanisms Are Required for Epidermal Growth Factor (EGF)-stimulated Extracellular Signal-regulated Kinase (ERK) Activity Pulses

In both physiological and cell culture systems, EGF-stimulated ERK activity occurs in discrete pulses within individual cells. Many feedback loops are present in the EGF receptor (EGFR)-ERK network, but the mechanisms driving pulsatile ERK kinetics are unknown. Here, we find that in cells that respond to EGF with frequency-modulated pulsatile ERK activity, stimulation through a heterologous TrkA receptor system results in non-pulsatile, amplitude-modulated activation of ERK. We further dissect the kinetics of pulse activity using a combination of FRET- and translocation-based reporters and find that EGFR activity is required to maintain ERK activity throughout the 10–20-minute lifetime of pulses. Together, these data indicate that feedbacks operating within the core Ras-Raf-MEK-ERK cascade are insufficient to drive discrete pulses of ERK activity and instead implicate mechanisms acting at the level of EGFR.     

Use of Cysteine Trapping to Map Spatial Approximations between Residues Contributing to the Helix N-capping Motif of Secretin and Distinct Residues within Each of the Extracellular Loops of Its Receptor

Amino-terminal regions of secretin-family peptides contain key determinants for biological activity and binding specificity, although the nature of interactions with receptors is unclear. A helix N-capping motif within this region has been postulated to directly contribute to agonist activity while also stabilizing formation of a helix extending toward the peptide carboxyl terminus and docking within the receptor amino terminus. We used cysteine trapping to systematically explore spatial approximations between cysteines replacing each residue in this motif of secretin (sec), Phe⁶, Thr⁷, and Leu¹⁰, and cysteines incorporated into the extracellular face of the receptor. Each peptide was a full agonist for cAMP, but had a lower binding affinity than natural hormone. These bound to COS cells expressing 61 receptor constructs incorporating cysteines in every position along each extracellular loop (ECL) and adjacent parts of transmembrane (TM) segments. Patterns of covalent labeling were distinct for each probe, with Cys⁶-sec labeling multiple residues in the carboxyl-terminal half of ECL2 and throughout ECL3, Cys⁷-sec predominantly labeling only single residues in the carboxyl-terminal end of ECL2 and the amino-terminal end of ECL3, and Cys¹⁰-sec not efficiently labeling any of these residues. These spatial constraints were used to refine our model of secretin bound to its receptor, now bringing ECL3 above the amino terminus of the ligand and revealing possible charge-charge interactions between this part of secretin and receptor residues in TM5, TM6, ECL2, and ECL3, which can orient and stabilize the peptide-receptor complex. This was validated by testing predicted approximations by mutagenesis and residue-residue complementation studies.     

Interactions of cyclic nucleotide-gated channel subunits and protein tyrosine kinase probed with genistein

The cGMP sensitivity of cyclic nucleotide-gated (CNG) channels can be modulated by changes in phosphorylation catalyzed by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases. Previously, we used genistein, a PTK inhibitor, to probe the interaction between PTKs and homomeric channels comprised of alpha subunits (RETalpha) of rod photoreceptor CNG channels expressed in Xenopus oocytes. We showed that in addition to inhibiting phosphorylation, genistein triggers a noncatalytic interaction between PTKs and homomeric RETalpha channels that allosterically inhibits channel gating. Here, we show that native CNG channels from rods, cones, and olfactory receptor neurons also exhibit noncatalytic inhibition induced by genistein, suggesting that in each of these sensory cells, CNG channels are part of a regulatory complex that contains PTKs. Native CNG channels are heteromers, containing beta as well as alpha subunits. To determine the contributions of alpha and beta subunits to genistein inhibition, we compared the effect of genistein on native, homomeric (RETalpha and OLFalpha), and heteromeric (RETalpha+beta, OLFalpha+beta, and OLFalpha+RETbeta) CNG channels. We found that genistein only inhibits channels that contain either the RETalpha or the OLFbeta subunits. This finding, along with other observations about the maximal effect of genistein and the Hill coefficient of genistein inhibition, suggests that the RETalpha and OLFbeta subunits contain binding sites for the PTK, whereas RETbeta and OLFalpha subunits do not.     

Human Epidermal Growth Factor Receptor 2 (HER2) Impedes MLK3 Kinase Activity to Support Breast Cancer Cell Survival

Human epidermal growth factor receptor 2 (HER2) is amplified in ∼15–20% of human breast cancer and is important for tumor etiology and therapeutic options of breast cancer. Up-regulation of HER2 oncogene initiates cascades of events cumulating to the stimulation of transforming PI3K/AKT signaling, which also plays a dominant role in supporting cell survival and efficacy of HER2-directed therapies. Although investigating the underlying mechanisms by which HER2 promotes cell survival, we noticed a profound reduction in the kinase activity of a pro-apoptotic mixed lineage kinase 3 (MLK3) in HER2-positive (HER2+) but not in HER2-negative (HER2−) breast cancer tissues, whereas both HER2+ and HER2− tumors expressed a comparable level of MLK3 protein. Furthermore, the kinase activity of MLK3 was inversely correlated with HER2+ tumor grades. Moreover, HER2-directed drugs such as trastuzumab and lapatinib as well as depletion of HER2 or HER3 stimulated MLK3 kinase activity in HER2+ breast cancer cell lines. In addition, the noted inhibitory effect of HER2 on MLK3 kinase activity was mediated via its phosphorylation on Ser⁶⁷⁴ by AKT and that pharmacological inhibitors of PI3K/AKT prevented trastuzumab- and lapatinib-induced stimulation of MLK3 activity. Consistent with the pro-apoptotic function of MLK3, stable knockdown of MLK3 in the HER2+ cell line blunted the pro-apoptotic effects of trastuzumab and lapatinib. These findings suggest that HER2 activation inhibits the pro-apoptotic function of MLK3, which plays a mechanistic role in mediating anti-tumor activities of HER2-directed therapies. In brief, MLK3 represents a newly recognized integral component of HER2 biology in HER2+ breast tumors.     

The Full-Length, Cytoplasmic C-Terminus of the β2-Adrenergic Receptor Expressed in E. coli Acts as a Substrate for Phosphorylation by Protein Kinase A, Insulin Receptor Tyrosine Kinase, GRK2, but Not Protein Kinase C and Suppresses Desensitization when Expressed in Vivo

The ability of the cytoplasmic, full-length C-terminus of the β2-adrenergic receptor (BAC1) expressed in Escherichia coli to act as a functional domain and substrate for protein phosphorylation was tested. BAC1 was expressed at high-levels, purified, and examined in solution as a substrate for protein phosphorylation. The mobility of BAC1 on SDS–PAGE mimics that of the native receptor itself, displaying decreased mobility upon chemical reduction of disulfide bonds. Importantly, the C-terminal, cytoplasmic domain of the receptor expressed in E. coli was determined to be a substrate for phosphorylation by several candidate protein kinases known to regulate G-protein-linked receptors. Mapping was performed by proteolytic degradation and matrix-assisted laser desorption ionization, time-of-flight mass spectrometry. Purified BAC1 is phosphorylated readily by protein kinase A, the phosphorylation occurring within the predicted motif RRSSSK. The kinetic properties of the phosphorylation by protein kinase A displayed cooperative character. The activated insulin receptor tyrosine kinase, which phosphorylates the beta-adrenergic receptor in vivo, phosphorylates BAC1. The Y364 residue of BAC1 was predominantly phosphorylated by the insulin receptor kinase. GRK2 catalyzed modest phosphorylation of BAC1. Phosphorylation of the human analog of BAC1 in which Cys341 and Cys378 were mutated to minimize disulfide bonding constraints, displayed robust phosphorylation following thermal activation, suggesting under standard conditions that the population of BAC1 molecules capable of assuming the “activated” conformer required by GRKs is low. BAC1 was not a substrate for protein kinase C, suggesting that the canonical site in the second cytoplasmic loop of the intact receptor is preferred. The functional nature of BAC1 was tested additionally by expression of BAC1 protein in human epidermoid carcinoma A431 cells. BAC1 was found to act as a dominant-negative, blocking agonist-induced desensitization of the beta-adrenergic receptor when expressed in mammalian cells. Thus, the C-terminal, cytoplasmic tail of this G-protein-linked receptor expressed in E. coli acts as a functional domain, displaying fidelity with regard to protein kinase action in vivo and acting as a dominant-negative with respect to agonist-induced desensitization.     

Presence of Platelet-Activating Factor and Its Receptor in Baboon (Papio spp) Spermatozoa

Platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF] is a novel potent signaling phospholipid which has unique pleiotropic biological properties in addition to platelet activation. PAF has been detected in the spermatozoa in several species. The concentration of PAF is inversely related to human spermatozoal quality. PAF is present in squirrel monkey (a seasonal breeder) spermatozoa and is significantly higher during the breeding season. PAFs mechanism of action is a receptor-mediated event. There is no report on the presence of PAF or the PAF-receptor in nonhuman Old World primate spermatozoa. Therefore, the primary objective of this study was to determine if PAF is present in the spermatozoa from baboons, which are year-round breeders. A secondary objective was to determine the presence and localization of the PAF-receptor in spermatozoa. We extracted endogenous lipids from mature hybrid baboon (Papio spp) epididymal spermatozoa and assayed them for the presence of PAF by [ ¹²⁵ I]-radioimmunoassay. We also exposed baboon spermatozoa to PAF-receptor antibody followed by FITC-conjugated antibody. PAF was in all samples assayed (mean: 2.29 (±0.63) pmol/10 ⁶ spermatozoa). Baboon spermatozoa possess PAF-receptors most prevalently along the neck and midpiece regions. The data demonstrates that PAF and its receptor are present in baboon spermatozoa. Additional studies will elucidate the role of PAF in spermatozoal function.     

Different Epidermal Growth Factor Receptor (EGFR) Agonists Produce Unique Signatures for the Recruitment of Downstream Signaling Proteins

The EGF receptor can bind seven different agonist ligands. Although each agonist appears to stimulate the same suite of downstream signaling proteins, different agonists are capable of inducing distinct responses in the same cell. To determine the basis for these differences, we used luciferase fragment complementation imaging to monitor the recruitment of Cbl, CrkL, Gab1, Grb2, PI3K, p52 Shc, p66 Shc, and Shp2 to the EGF receptor when stimulated by the seven EGF receptor ligands. Recruitment of all eight proteins was rapid, dose-dependent, and inhibited by erlotinib and lapatinib, although to differing extents. Comparison of the time course of recruitment of the eight proteins in response to a fixed concentration of each growth factor revealed differences among the growth factors that could contribute to their differing biological effects. Principal component analysis of the resulting data set confirmed that the recruitment of these proteins differed between agonists and also between different doses of the same agonist. Ensemble clustering of the overall response to the different growth factors suggests that these EGF receptor ligands fall into two major groups as follows: (i) EGF, amphiregulin, and EPR; and (ii) betacellulin, TGFα, and epigen. Heparin-binding EGF is distantly related to both clusters. Our data identify differences in network utilization by different EGF receptor agonists and highlight the need to characterize network interactions under conditions other than high dose EGF.     

Time-Resolved Signaling Pathways of Nerve Growth Factor Diverge Downstream of the p140trk Receptor Activation Between Chick Sympathetic and Dorsal Root Ganglion Sensory Neurons

Abstract: We have recently shown that the small GTP binding protein p21 ras is essential for nerve growth factor (NGF)-mediated survival of peripheral embryonic chick dorsal root ganglia (DRG) sensory but not sympathetic neurons. To investigate at which level of the signaling cascade the pathways diverge, we have studied the time-resolved pattern of NGF-stimulated tyrosine phosphorylation of proteins within 4 h after addition of the neurotrophin. In both chick sympathetic neurons [embryonic day (E) 12] and DRG sensory neurons (E9) NGF induces within 1 min the autophosphorylation of the receptor tyrosine kinase p140trk. However, the pattern of substrate protein tyrosine phosphorylation downstream of p140trk is distinctly different in both neuronal subtypes. In sympathetic neurons, we observe within 1 min the tyrosine phosphorylation of a new substrate protein, p105, reaching maximal levels at 3 min. Tyrosine phosphorylation of p105 remains elevated for up to 4 h. Subsequent to p105, NGF induces the tyrosine phosphorylation of p42, a protein belonging to the family of mitogen-activated protein (MAP) kinases. This stimulation is transient, reaching maximal levels at 10 min and returning to very low levels already after 2 h. In DRG sensory neurons, tyrosine phosphorylation of p105 is weak and very short lived, disappearing already after treatment with NGF for 10 min. In contrast, activation of MAP kinase p42 in DRG sensory neurons is more stable than in sympathetic neurons. All NGF-stimulated tyrosine phosphorylation events were inhibited by preincubation of neurons with the tropomyosin-related kinase (trk) inhibitor K252a. We suggest the working hypothesis that persistent tyrosine phosphorylation of p105 may play a role in the p21ras-independent NGF survival pathway of chick sympathetic neurons.     

Binding Studies of TNF Receptor Superfamily (TNFRSF) Receptors on Intact Cells

Ligands of the tumor necrosis factor superfamily (TNFSF) interact with members of the TNF receptor superfamily (TNFRSF). TNFSF ligand-TNFRSF receptor interactions have been intensively evaluated by many groups. The affinities of TNFSF ligand-TNFRSF receptor interactions are highly dependent on the oligomerization state of the receptor, and cellular factors (e.g. actin cytoskeleton and lipid rafts) influence the assembly of ligand-receptor complexes, too. Binding studies on TNFSF ligand-TNFRSF receptor interactions were typically performed using cell-free assays with recombinant fusion proteins that contain varying numbers of TNFRSF ectodomains. It is therefore not surprising that affinities determined for an individual TNFSF ligand-TNFRSF interaction differ sometimes by several orders of magnitude and often do not reflect the ligand activity observed in cellular assays. To overcome the intrinsic limitations of cell-free binding studies and usage of recombinant receptor domains, we performed comprehensive binding studies with Gaussia princeps luciferase TNFSF ligand fusion proteins for cell-bound TNFRSF members on intact cells at 37 °C. The affinities of the TNFSF ligand G. princeps luciferase-fusion proteins ranged between 0.01 and 19 nm and offer the currently most comprehensive and best suited panel of affinities for in silico studies of ligand-receptor systems of the TNF family.     

Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling

Arginine methylation is a common post-translational modification, but its role in regulating protein function is poorly understood. This study demonstrates that, TNF receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase involved in innate immune signaling, is regulated by reversible arginine methylation in a range of primary and cultured cells. Under basal conditions, TRAF6 is methylated by the methyltransferase PRMT1, and this inhibits its ubiquitin ligase activity, reducing activation of toll-like receptor signaling. In response to toll-like receptor ligands, TRAF6 is demethylated by the Jumonji domain protein JMJD6. Demethylation is required for maximal activation of NF-κB. Loss of JMJD6 leads to reduced response, and loss of PRMT1 leads to basal pathway activation with subsequent desensitization to ligands. In human primary cells, variations in the PRMT1/JMJD6 ratio significantly correlate with TRAF6 methylation, basal activation of NF-κB, and magnitude of response to LPS. Reversible arginine methylation of TRAF6 by the opposing effects of PRMT1 and JMJD6 is, therefore, a novel mechanism for regulation of innate immune pathways.     

表皮生长因子受体酪氨酸激酶结构域与其抑制剂染料木素的模拟对接

染料木素是表皮生长因子受体酪氨酸激酶结构域(EGFR-TK)高度特异的非竞争性抑制剂.本研究采用AUTODOCK3.05分子对接软件包对EGFR-TK与染料木素进行了模拟对接研究,探究了二者的相互作用机制,为染料木素的抗肿瘤机制提供理论依据.对接结果表明,染料木素结合在EGFR-TK的活性腔中,与EGFR-TK发生了强烈的相互作用,结合自由能△G为-31.2 kJ/mol;染料木素通过干扰TK催化活性结构中Lys721/Glu738离子对的形成而抑制了EGFR-TK的活性,属于非竞争性结合和抑制作用;在结合中,疏水力和氢键发挥了重要作用.     

Three-dimensional Model Structure for the Extracellular Domains of Fibroblast Growth Factor Receptor - 1 (FGFR-1)

We have developed a model for the two immunoglobulin-like extracellular domains DII and DIII of the FGF receptor 1 (FGFR-1), giving a special attention to the determination of the appropriate Ig set. The DII domain was aligned with the C-terminal domain of myosin light chain kinase (telokin) of the I set, and the DIII domain with the variable domain of the Bence-Jones immunoglobulin of the V set. Two assemblies, corresponding to different propositions for the domains relative orientation, have been refined and compared.Electronic Supplementary Material available.     

K-562细胞胰岛素受体酪氨酸蛋白激酶活性及内源性底物的研究

本文对丁酸钠诱导分化的人白血病细胞株K-562细胞的胰岛素受体酪氨酸蛋白激酶性及细胞内源性底物进行了研究。结果表明,诱导分化后的细胞酪氨酸蛋白激活酶活性降低,胰岛素受体数量减少,酪氨酸蛋白激酶的一底物蛋白在分化后消失。该研究结果及其在该方面的进一步研究有可能为白血病发病机制的阐明以及为临床治疗白血病开辟靳途径提供一些有价值的线索。     

Design,construction, crystallization,and preliminary X-ray studies of a fine-tuning mutant (F133V) of module-substituted chimera hemoglobin

A chimera βα-subunit of human hemoglobin was crystallized into a carbonmonoxy form. The protein was assembled by substituting the structural portion of a β-subunit of hemoglobin (M4 module of the subunit) for its counterpart in the α-subunit. In order to overcome the inherent instability in the crystallization of the chimera subunit, a site-directed mutagenesis (F133V) technique was employed based on a computer model. The crystal was used for an X-ray diffraction study yielding a data set with a resolution of 2.5 Å. The crystal belongs to the monoclinic space group P2₁, with cell dimensions of a = 62.9, b = 81.3, c = 55.1 Å, and β = 91.0°. These dimensions are similar to the crystallographic parameters of the native β-subunit tetramers in three different ligand states, one of which is a cyanide form that was also crystallized in this study. Proteins 32:263–267, 1998. © 1998 Wiley-Liss, Inc.     

Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors

Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional “three-finger” snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by “three-finger” snake neurotoxins.     

A kinetic model of phospholipase C-γ1 linking structure-based insights to dynamics of enzyme autoinhibition and activation

Phospholipase C-γ1 (PLC-γ1) is a receptor-proximal enzyme that promotes signal transduction through PKC in mammalian cells. Because of the complexity of PLC-γ1 regulation, a two-state (inactive/active) model does not account for the intricacy of activation and inactivation steps at the plasma membrane. Here, we introduce a structure-based kinetic model of PLC-γ1, considering interactions of its regulatory Src homology 2 (SH2) domains and perturbation of those dynamics upon phosphorylation of Tyr783, a hallmark of activation. For PLC-γ1 phosphorylation to dramatically enhance enzyme activation as observed, we found that high intramolecular affinity of the C-terminal SH2 (cSH2) domain–pTyr783 interaction is critical, but this affinity need not outcompete the autoinhibitory interaction of the cSH2 domain. Under conditions for which steady-state PLC-γ1 activity is sensitive to the rate of Tyr783 phosphorylation, maintenance of the active state is surprisingly insensitive to the phosphorylation rate, since pTyr783 is well protected by the cSH2 domain while the enzyme is active. In contrast, maintenance of enzyme activity is sensitive to the rate of PLC-γ1 membrane (re)binding. Accordingly, we found that hypothetical PLC-γ1 mutations that either weaken autoinhibition or strengthen membrane binding influence the activation kinetics differently, which could inform the characterization of oncogenic variants. Finally, we used this newly informed kinetic scheme to refine a spatial model of PLC/PKC polarization during chemotaxis. The refined model showed improved stability of the polarized pattern while corroborating previous qualitative predictions. As demonstrated here for PLC-γ1, this approach may be adapted to model the dynamics of other receptor- and membrane-proximal enzymes.